1. Field of the Invention
The present invention relates to a method of preparing 2-deoxyribose 5-phosphate stably at a high yield by using a microorganism itself or an enzyme derived from the microorganism. The present invention also relates to a microorganism capable of producing an enzyme which can be utilized for the preparation of 2-deoxyribose 5-phosphate. 2-deoxyribose 5-phosphate is used as a starting material in the biochemical synthesis of deoxynucleosides. Further, by dephosphorylating 2-deoxyribose 5-phosphate, 2-deoxyribose can be obtained. 2-deoxyribose is useful as a starting material in the chemical synthesis of nucleosides.
2. Description of the Related Art
2-deoxyribose 5-phosphate has conventionally been prepared by hydrolyzing DNA with an enzyme or chemically phosphorylating 2-deoxyribose. However, the former method has a problem that DNA as the raw material is expensive and a number of separation/purification processes are required. The latter method also has a problem that regioselective phosphorylation of 2-deoxyribose is difficult. Thus, 2-deoxyribose 5-phosphate cannot be prepared inexpensively by either of the above-mentioned two methods.
In vivo, it has been known that 2-deoxyribose 5-phosphate is produced from glyceraldehyde 3-phosphate and acetaldehyde, by the catalytic action of 2-deoxyribose-5-phosphate aldolase (deoxyribose-phosphate aldolase EC 4.1.2.4). However, the preparation of 2-deoxyribose 5-phosphate according to the aforementioned reaction has a problem that chemical synthesis of glyceraldehyde 3-phosphate as one substrate is not easy and glyceraldehyde as the other substrate is unstable and apt to be isomerized to dihydroxyacetone that is a more stable isomer.
It is also known that glyceraldehyde 3-phosphate is produced in vivo, as a result of an isomerization reaction in which dihydroxyacetone phosphate is isomerized by triose-phosphate isomerase (EC 5.3.1.1). Dihydroxyacetone phosphate as the substrate of the aforementioned reaction can be chemically or biochemically synthesized (refer to, for example, Itoh, N., Tsujibata, Y., Liu, J. Q., Appl. Microbiol. Biotechnol., volume 51, pp. 193-200, 1999).
However, the object of the aforementioned reports is academic analysis of the in vivo metabolism of pentose and the stereospecific action of aldolase, and the industrial production of glyceraldehyde 3-phosphate and 2-deoxyribose 5-phosphate is utterly beyond the scope of the reports. Up to now, a method of industrially producing 2-deoxyribose 5-phosphate has not been reported.
Regarding the preparation of 2-deoxyribose 5-phosphate by using an enzyme or an enzyme reagent, several examples have been reported in academic literatures as follows.
As one of these examples, there exists a report that 2-deoxyribose 5-phosphate was produced from glyceraldehyde 3-phosphate and acetaldehyde as the substrates, by the action of 2-deoxyribose-5-phosphate aldolase (Barbas, III, C. F., Wang, Y., Wong, C., J. Am. Chem. Soc., vol. 112, pp. 2013-2014, 1990). However, the production yield of 2-deoxyribose 5-phosphate with respect to the amount of the substrates as the raw materials cannot be known in this report, because the report does not disclose the amount of produced 2-deoxyribose 5-phosphate.
Further, as another example, there exists a report that 2-deoxyribose 5-phosphate was obtained from dihydroxyacetone phosphate and acetaldehyde as the substrates, by using a commercially available triose-phosphate isomerase as a biochemical reagent and a 2-deoxyribose-5-phosphate aldolase crude enzyme prepared from Escherichia coli which had been transformed with a plasmid having 2-deoxyribose-5-phosphate aldolase gene (deo C gene), in the presence of EDTA as a phosphatase inhibitor and nitrogen gas (Chen, L, Dumas, D. P., Wong, C., J. Am. Chem. Soc., vol. 114, pp. 741-748, 1992). However, in these academic reports, the enzymes are derived from different origins and purified at the level of a reagent. In addition, the influence of phosphatase cannot be completely eliminated, though a significant amount of EDTA is used in order to inhibit dephosphorylation by phosphatase. For this reason, the method is not suitable for industrial production of 2-deoxyribose 5-phosphate.
Glyceraldehyde 3-phosphate is an important intermediate in the saccharometabolism such as glycolytic pathway and pentose phosphate cycle (refer to, for example, page 411, the third edition, “Seikagaku Jiten (Dictionary of Biochemistry)”, 1998, Tokyo Kagaku Dojin). Accordingly, glyceraldehyde 3-phosphate is metabolized to various courses by various enzymes in a cell. Also, there is a problem that the phosphate group of glyceraldehyde 3-phosphate tends to be easily cut off by phosphatase.